Intracellular delivery of drugs, proteins and genes into viable cells in one of the greatest challenges in drug and gene delivery. Previous studies show that exposure of cells to ultrasound under appropriate conditions drives molecules into cells to non-invasively increase pharmacological effect of drugs and expression of genes in a variety of different cell types in vitro and in animals in vivo. These effects occur under different ultrasound conditions from those employed in clinical ultrasonic imaging or heating. In the context of this new use of ultrasound, the mechanisms are not known by which compounds can be driven into cells and, as a common side effect, some cells can be killed in the process. Our previous work in this area has emphasized the acoustic and physical conditions that deliver molecules into viable cells. For this reason, the Specific Aims of this proposal address biological and biophysical mechanisms of the cell's response by determining (1) the mechanism(s) by which molecules are taken up into cells exposed to ultrasound and (2) the mechanism(s) by which ceils die when exposed to ultrasound. Carrying out these Aims will make significant advances toward the long term goal of rationally designing protocols, formulations and devices that achieve high levels of intracellular delivery while maintaining high cell viability. Specifically, Aim 1 studies are guided by the hypothesis that molecular uptake occurs by diffusion through membrane disruptions on the order of 1 micron in size that reseal over a lifetime on the order of 1 minute by active patching using intracellular vesicles. Experiments will determine the existence, size and lifetime of membrane disruptions, as well as the mechanism by which disruptions are actively or passively resealed. Transport through membrane disruptions will be modeled mathematically and the role of active transport will be examined too. Aim 2 studies are guided by the hypothesis that cell death can occur on a timescale of seconds and exhibits characteristic features of apoptosis, necrosis and paraptosis. Experiments will identify characteristic nuclear, mitochondrial, ultrastructural and enzymatic features of apoptosis and contrast them with features of necrosis and paraptosis. The kinetics of cell death is of special interest, since preliminary results suggest that cells exhibit characteristics of late stage apoptosis within seconds after exposure to ultrasound, which contrasts with kinetics typically of hours observed by other apoptosis mechanisms. Studies will employ multiple forms of electron and confocal microscopy, flow cytometry and mathematical analysis as their core tools guided in part by comparison with known mechanisms of cell membrane disruption, including electroporation and mechanical cell wounding. During the final year, mechanistic findings will be used to design and test ultrasound protocols that optimize molecular uptake and cell viability.